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Secondary structure of the MutT enzyme as determined by NMR
D J Weber1, C Abeygunawardana, M J Bessman
1Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Abstract:
The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
The MutT enzyme
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- The MutT enzyme is crucial for hydrolyzing nucleoside triphosphates (NTPs).
- Previous studies established NMR assignments for MutT, providing a foundation for structural analysis.
Purpose of the Study:
- To determine the secondary structure of the MutT enzyme in solution.
- To characterize the helical and beta-sheet content of MutT using NMR data.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy, including 2D and 3D homonuclear and heteronuclear experiments.
- Analysis of Nuclear Overhauser Effect (NOE) data and amide NH exchange rates.
- Comparison of NMR-derived helical content with circular dichroism spectroscopy results.
Main Results:
- MutT possesses two alpha-helices (residues 47-59 and 119-128) and a mixed parallel/antiparallel beta-sheet with five strands (residues 3-13, 18-24, 70-74, 79-87, and 102-106).
- NMR-predicted helical content (17.8%) aligns with circular dichroism measurements (20.9%).
Conclusions:
- The study elucidates the secondary structure of the MutT enzyme in solution.
- The findings provide critical insights into the structural basis of MutT's enzymatic activity.